The art of liquid column chromatography is an old and well known means for separating a material from a sample. Depending upon the sample and materials to be separated therefrom, one of a variety of modes of liquid column chromatography is used to effect the separation. Such chromatographic modes include size exclusion (SEC), ion-exchange, reversed phase, normal phase, hydrophobic interaction, hydrophilic interaction, affinity, donor-acceptor, ion-pair and chiral separation chromatography.
Size-exclusion chromatography (SEC) generally comprises the differential elution of solutes from a bed of a porous chromatographic medium, caused by different degrees of steric exclusion of the solute molecules from the pore volume in pores with smaller size than is the size of the molecule to be eluted. The smaller the molecule, the more pores (the higher pore volume) of the porous separation medium (column packing) is available for their penetration. SEC is used in the separation of macromolecules according to their actual size defined by their hydrodynamic volume. Ideally, there is no interaction of any kind between the solute molecules and the chromatographic separation medium in SEC as the separation is driven by entropic changes.
In contrast to SEC, all other modes of liquid chromatography are based on an interaction between the compounds to be separated dissolved in a mobile phase and the stationary phase, i.e. the solid column packing which is why these modes are called interactive. The driving force of the separation is the difference in enthalpy of interactions of different compounds.
Classical column liquid adsorption chromatography, also referred to as normal or direct phase chromatography, is performed on a hydrophilic adsorbent such as silica and alumina with non-polar to moderately polar solvents. Normal phase chromatography has many drawbacks and the frequency of its use is declining.
Reversed phase chromatography (RPC) has been the most important branch of high-performance liquid chromatography (HPLC) since the early 1970s. The system for RPC consists of nonpolar (hydrophobic) stationary phase and a polar mobile phase. The primary interaction responsible for retention is essentially a solvent effect similar to the hydrophobic effect. Specifically, it is a noncovalent association of non-polar moieties in aqueous media. As the interactions depend on the type of molecules, they are the driving force for the separation. The typical mobile phases used in RPC are aqueous solutions of displacement agents, such as acetonitrile or 1-propanol. Elution with a mobile phase having constant composition (isocractic elution) is typically used in separations in low molecular weight compounds, while large molecules, like proteins, are eluted with a mobile phase in which the concentration of the displacement agent increases gradually (gradient elution).
Hydrophobic interaction chromatography (HIC) is an important separation mode for purification and separation of biomolecules. In this mode, hydrophobic ligands are chemically attached to hydrophilic matrix and a distinct interaction is obtained between biomolecules, such as proteins, and the stationary phase surface in the presence of high concentration of antichaotropic salt aqueous solution at neutral pH. Elution is achieved by diminishing the hydrophobic interaction by a descending salt gradient.
Hydrophilic interaction works in a similar manner as RPC except that the porous separation medium contains hydrophilic groups instead of hydrophobic groups.
Ion-exchange, in a broad sense, is the reversible interchange of ions of like charge between a solution and a solid, insoluble material in contact with it, the ion exchanger. All ion exchanges are reversible, but the equilibria for different ions under particular conditions vary widely, and it is these variations that make ion-exchange chromatography possible. The ion exchange is controlled by electrostatic interaction between the ions being exchanged, the mobile ions in a solution, and the fixed ions either acidic groups (carboxyl, sulfonate) or basic groups (tertiary and quaternary amines).
Ion chromatography is a separation of ionic species, typically low molecular weight anions or cations, on a column packed with a low capacity ion exchanger with detection by electrical conductivity. The ions to be separated are retained in the column according to the strength of their interaction with attached ion-exchange groups in very dilute eluent.
Ion-exchange chromatography is often used for separation of proteins and other large charged biopolymer molecules. The proteins are adsorbed into the separation medium at the beginning of the separation process in a mobile phase buffer with low ionic strength. The elution of individual components of the separated mixture is achieved with an increasing salt concentration gradient in the mobile phase.
An alternative to ion-exchange chromatography for analysis of organic anions, like those of alkaloids, peptides, or surfactants, is ion-pair chromatography. The anions combine with a cationic surfactant, such as cetyltrimethylammonium bromide, to form hydrophobic complexes which are separated in a standard reversed phase chromatography.
Ligand-exchange chromatography depends on the exchange of electron-donor ligand around a central metal ion loaded in a special cation exchanger. The metal ion does not move while the ligand(s) coordinated to it is exchanged according to its complex building ability. The most exciting application of ion-exchange chromatography is the separation of optical isomers of amino acids. The chiral resolving ligand, such as L-proline, is attached to the stationary phase and a copper complex is made. The D-form of the amino acid binds more tightly to the solid phase while the L-form is eluted already with water and very good separation is achieved.
Separation of chiral molecules based on enantioselective adsorption may also be achieved upon another mechanism other than ligand exchange. The chiral recognition and the retention is controlled by hydrogen binding, .pi.-.pi. interaction. The separation medium plays a very important role in chiral separation chromatography and it has to be perfectly designed to contain at least three points of interaction between chiral separation phase and analyte molecule, at least two of which should be attractive.
The application of the donor-acceptor complex formation results in the donor-acceptor complex chromatography (DACC). A donor or an acceptor is chemically bonded to the surface of an insoluble porous matrix and separates solutes possessing acceptor or donor properties, respectively. Typical electron acceptor phases contain attached dinitroanilinoalkyl groups while the typical electron donor phase is featured by pentamethylphenyl or phenanthryl groups. The mobile phase is non-polar but, surprisingly, the media work in the polar mobile phase as well. The major application area of DAAC is separation of unsaturated organic compounds, chlorinated aromatic coumpounds, amino acids, on one hand, and polynitro-substituted aromatic compounds and similar derivatives, on the other. DAAC approach is also useful for the separation of enantiomers.
Affinity chromatography incorporates a large family of adsorption chromatography methods, all of which utilize more or less specific interaction between biological molecules in solution and covalently attached ligand molecules on a solid phase. In addition to classical biospecific affinity chromatography, the methods are charge-transfer affinity chromatography (similar to DACC), immobilized metal affinity chromatography (similar to ligand exchange chromatography), dye ligand affinity chromatography, immunoaffinity chromatography (immunosorption), and covalent chromatography (chemisorption). The names specify usually the immobilized ligand or type of interaction. The higher the specificity of the solute-sorbent interaction, the closer the separation process amounts to "fishing out" the particular biological molecule, and the further the process is from the typical chromatographic separation. The most specific immuno-affinity chromatography based on interaction with an immobilized antibody results in the capture of one single antigen dissolved in the mobile phase without requiring the separation of all other components of the sample. After saturation of the column capacity with the soluble antigen, the adsorbed column is washed free of any contaminant compounds and the antigen is displaced from the solid phase of the separation medium. The separation may be repeated again after re-equilibration of the column.
Each of these chromatographic modes is particularly useful for separations of specific groups of compounds. The separation media are specially designed for a particular chromatographic mode and usually do not work in another mode adequately.
The problem with such specific separation techniques is that often a liquid sample contains a variety of molecules which require different modes of separation. This, however, is not easily accomplished because different modes of separations usually require different separation media to effect the separation of different molecules. This requires the use of multiple columns and multiple separation media to accomplish the desired separations.
It may be possible to use combinations of different separation media in different columns for multimodal separations. An example of this multiple column bimodal separation was described recently by Wheatley J. B., J. Chromatogr., 603 (1992) 273. The bimodal separation of small molecules in one column packed with one separation medium and based on sequential multimodal elution was described by Little E. L., Jeansonne M. S., Foley J. P.; Anal Chem., 63, 1991, 33. They combined ion-exchange and reversed phase chromatography for the separation of a complex sample containing two groups of compounds: charged and non-polar. The use of two different gradients, i.e. a pH gradient and a methanol gradient, resulted in the separation of the charged molecules first, followed by the separation of the neutral molecules after switching to the second mobile phase. This approach makes use of imperfect surface functionalization of porous silica beads which contained C.sub.1, C.sub.8 or C.sub.18 groups together with the original acidic surface silanol groups. Similarly, the DIONEX OmniPack PAX-500 column is packed with non-porous poly[styrene-divinylbenzene] beads coated on the bead surface with attached ion-exchange latex particles (as described by the DIONEX booklet). Here again, the coating of the bead surface is imperfect and it is the non-covered hydrophobic areas of the original non-porous beads that are used for separation in the second mode. This approach excludes combinations not involving the reversed phase mode (the original ST-DVB surface remains non-polar even after attachment of latex particles) as well as any size exclusion separation.
These prior bimodal separations fail when used with a large number of biological samples where biopolymers, like proteins or nucleic acids, are present along with small molecules, such as drugs, metabolites, pollutants, exo- and endotoxins, etc. Since sample pretreatment, like solvent extraction, solid phase extraction or ultrafiltration, is time-consuming and tedious, new stationary phases have been developed which prevent contact between the groups used for separation in the reversed phase or ion-exchange chromatography. Media that have very small pores preventing large molecules from penetration into the bead (total exclusion), are mostly used. Clogging of the column by proteins stuck on the bead surface is inhibited by providing the surface with hydrophilic groups. This approach was reviewed recently (Pinkerton T. C., J. Chromatogr., 544, (1991) 13; Haginaka J., Trends Anal. Chem., 10, (1991) 17). It may be called pseudomultimodal as one of the modes is actually not a chromatography but a simple filtration-like separation (total exclusion of all molecules exceeding a size limit). The separation of individual components of the excluded part of the sample, however, requires an additional column. Moreover, such bimodal separation as disclosed by Foley et al and Dionex are not generally useful but rather are applicable solely to the specific modes of separation disclosed.
It would be a substantial advantage to develop a process which could use different combinations of the various modes of chromatographic separation depending upon the molecules to be separated without changing the separation medium within the column or using different columns. Such a process would be more economical and time efficient.